Morphological diversity, phenolic acids, and antioxidant properties in eryngo (Eryngium caucasicum Trautv): Selection of superior populations for agri‐food industry

Abstract Eryngo (Eryngium caucasicum Trautv) a widespread species of the Apiaceae reveals high nutritional value and therapeutic properties due to the significant content of biologically active metabolites such as essential oils, phenolic compounds, and flavonoids. The present study was performed to evaluate the morphological and biochemical variability and antioxidant properties of naturally grown populations of eryngo. One‐way ANOVA showed significant (p < .01) differences in the majority of parameters measured among the studied populations. The range of fresh weight was from 1.3 to 12.0 g/plant, while dry weight varied from 0.01 to 6.0 g/plant. The highest variation was observed for essential oil yield (CV = 205.32%) followed by essential oil content (CV = 126.23%) and chicoric acid content (CV = 71.18%). Total phenolics content varied from 8.85 to 88.15 mg GAE/g extract. Total flavonoids value ranged from 5.41 to 134.40 mg QE/g extract. Rosmarinic acid and chicoric acid contents varied from 0.118–1.234 and 0.014–0.597 μg/g DW, respectively. DPPH free radical scavenging activity varied from 76.12 to 513.5 μg/mL, while it ranged from 156.7 to 477.1 μg/mL with the ferrous ions (Fe2+) chelating assay. Rosmarinic acid and chicoric acid showed a significant and positive correlation (r 0.01 = 0.81 and r 0.05 = 0.40) with total phenolics, respectively. The Ward dendrogram analysis revealed two different clusters based on the parameters measured, confirming high morpho‐phytochemical variability among the individuals and populations. Principal component analysis (PCA) revealed eight PCs which contributed to 99.97% of the overall variance, and leaf length, essential oil content, and antioxidant activity in terms of DPPH and Fe2+ chelating techniques were the most effective attributes for characterizing and selecting the studied population. Based on the traits related to vegetative yield and antioxidant properties, eight individuals from two populations were superior for breeding and/or farming programs.


| INTRODUC TI ON
The genus Eryngium L. with more than 250 species is the largest in the family Umbelliferae (Apiaceae). It is broadly distributed throughout temperate areas of every continent. The Eryngium spp. are naturally grow in Central Asia, North and South America, North Africa, Australia, and Eurasia (Pimenov and Leonov, 1993;Calviño et al., 2008;Cortés-Fernández et al., 2022). In the wild flora of Iran, there are nine native species of Eryngium (Ghahreman 1997), of which eryngo (Eryngium caucasicum Trautv) is one of the most common and widespread species. It is a perennial herbaceous plant that has been distributed in Caucasia and North Eastern Anatolia across Northern, Middle Asia, Southern Russia, Eastern and Central Iran, Afghanistan, Pakistan, and Western Himalaya (Khoshbakht et al., 2007;Wörz 2004;Behmanesh et al., 2019). Due to its constantly increasing demand, E. caucasicum was introduced as a new crop plant in household gardens around the Caspian Sea and Alborz mountains in northern Iran.
Like the other plants of this family, Eryngium has been widely used as food, ornamental, vegetable, and/or in traditional medicine locally or worldwide (Zhang et al., 2008). The shoot parts of E. caucasicum are often used as a cooked vegetable, and/or as an aroma additive in the preparation of many traditional foods (Khoshbakht et al., 2007). Its fragrant young leaves are used as a stuffing mix in fish and chicken, and the cooked leaves are also consumed in soup or with yogurt (Eslami et al., 2011).
The chemical composition of E. caucasicum mainly consists of phenols, flavonoids, coumarins, and essential oils, presenting various redox capacities and pharmacological properties (Hashemabadi et al., 2010;Eslami et al., 2011;Behmanesh et al., 2019). Among the various plant secondary metabolites, phenolic compounds are of the most varied groups that have important health benefits, as they are believed to be the main factor for reducing cardiovascular disease risk in communities that use phenolics-rich foods and dietary supplements (Leopoldini et al., 2011;Sukhadiya et al., 2021). In addition, they play a vital role in plant adaptation/and or acclimation to various environmental perturbations.
Plant growth-related parameters, secondary metabolites biosynthesis, and their biological properties are influenced by environmental stresses, growth stimulants, and geographical and genetic factors (Sampaio et al., 2016;Ghorbanpour et al., 2016;Selmar et al., 2017;Meftahizadeh et al., 2019;Pourhosseini et al., 2020;Mohammadi et al., 2021;Mirheidari et al., 2022). Previous studies have shown the importance of certain morphological and biochemical traits regarding the productivity of plants in the Apiaceae family (Ninou et al., 2017;Adelifar et al., 2020;Jorkesh et al., 2020;). However, little is known about the phenotypic diversity and biochemical variation of E. caucasicum in different natural habitats. Thus, this study aimed to evaluate the morphological diversity and biochemical variation as well as antioxidant capacity in E. caucasicum populations from the northern parts of Iran.

| Plant materials and collection sites
In the present study, 60 naturally grown individuals of eryngo (Eryngium caucasicum Trautv) were sampled from different habitats/ populations of the Mazandaran (Javaherdeh, Dohezar, Sehezar, and Kelardasht) and Guilan (Javaherdashte and Eshkevar) provinces in the north of Iran. The distribution map of the studied populations is shown in Figure 1, and the climatic and geographical characteristics corresponding to these areas are presented in Table 1. The aerial parts/leaf materials were harvested during the growing season July-August 2019. Figure 2 F I G U R E 1 Map of the collection areas, Mazandaran (Dohezar, Sehezar, Javaherdeh, and Kelardasht) and Guilan (Eshkevar and Javaherdasht) for the studied populations of eryngo (Eryngium caucasicum Trautv) from north of Iran (the geographic and climatic characteristics of these locations are visible in Table 1)

| Morphological analysis
The key morphological parameters such as leaf size (length and width) and petiole length were determined using a digital caliper.
The fresh and dry weights of aerial parts were measured using a precision electronic balance in the range 0.001-1 g. In addition, the number of leaves/branches per plant was recorded manually. For these parameters, data were collected from 10 randomly selected individual plants in each population and their mean values were considered for statistical analysis.

| Biochemical analysis
The shoot parts of all collected samples were air-dried in the shade at ambient temperature (22-25°C) until constant weight and were considered for further laboratory/biochemical analyses.

| Essential oils isolation
For essential oils isolation, 30 g of the coarsely ground plant materials was hydrodistillated in a Clevenger-type apparatus using 500 ml of distilled water for approximately 3 h per sample based on the method quoted in British Pharmacopoeia (1998). After measuring the essential oil content (expressed as percentage w/w), the essential oil yield was calculated as dry weight × essential oil content of the sample (expressed as g/plant) (Singh et al., 2013).

| Measurement of total phenolics and flavonoids
Total phenolic content of the collected samples was measured using the Folin-Ciocalteu reagent with reference/standard compound, gallic acid (GA), following the procedure of Meda et al. (2005), and the final data were expressed as milligram of GAE per gram of dried extract.
The spectrophotometric assay was used to determine total flavonoid content based on aluminum chloride with quercetin (Q) as standard following the procedure of Chang et al. (2002). The obtained data were expressed as milligrams of QE per gram of dried extract.

| HPLC analysis for identification of phenolic compounds
In order to identify phenolic compounds of E. caucasicum samples, the (HPLC). Briefly, in a conical flask, 0.5 g of dried E. caucasicum powder was weighed and mixed with 10 ml of 80% methanol solution, followed by sonication at 40°C for 60 min. After initial centrifugation at 12,000 g for 1 h, the supernatants were filtered via 0.22 μm PEs membrane filter, and the filtrate was used as the test solution.
Then, 10 μl of test solution was injected into an U.S. Waters Alliance

HPLC system, equipped with the Uv 2487 Dual ʎ Absorbance
Detector and the C 18 column, with dimensions of 4.6 mm × 150 mm and 3.5 mm, with Millennium 32 software for analysis on an HPLC chromatography.

| DPPH scavenging assay
The antiradical activity of the collected samples was assessed based on DPPH radical scavenging activity following the protocol explained by Brand-Williams et al. (1995). The scavenging activity of DPPH was estimated through the following equation (Okoh et al., 2014).

| Statistical data analysis
Data were subjected to a one-way ANOVA test using SAS (SAS Institute,). The normality of all data was preliminary checked using the Shapiro-Wilk normality test. Relationships among the F I G U R E 2 Variability of morphological traits in eryngo (Eryngium caucasicum Trautv) populations studied individuals/and populations were evaluated by principal component analysis (PCA) using SPSS® software. Cluster analysis (UPGMA) was performed by the Euclidean distance method using PAST software.
Antioxidant measurements were done in triplicate

| Morphological variations
One-way ANOVA results showed a statistically-significant (p < .01) difference in all traits measured among the studied populations.
Therefore, it was possible to select the E. caucasicum populations for different values of a trait. The highest variation was observed for plant dry weight (CV = 81.41%) followed by leaf width (CV = 66.60%) and petiole length (CV = 52.17%). In contrast, the number of leaves/ branches per plant and leaf length showed the lowest CVs, 38.75% and 42.13%, respectively ( Table 2).
The most important traits of eryngo in food industries and various local foods are related to leaf growth and vegetative yield (Khoshbakht et al., 2007). Due to the photosynthesis process and subsequent secondary metabolites biosynthesis, leaf dimension and traits involving biomass are the most important parameters to be considered in agronomy and breeding/genomics programs (Ievinsh et al., 2020;Li et al., 2021).
Here, leaf length varied from 1.0 to 42.0 mm and leaf width ranged from 1.0 to 18.0 mm. Furthermore, petiole length varied from 12.0 to 96.0 mm, and the number of leaves/branches ranged from 1.0 to 3.0 ( Table 2). The range of fresh weight was from 1.3 to 12 g per plant, while dry weight varied from 0.01 to 6 g per plant with an average of 1.93 g/plant. The majority of individuals within the populations showed medium plant dry yield (35 individuals, 58.30%) followed by a high dry matter (5 individuals, 12.00%). Little is known about the growth habitat, phenotypic diversity, and biomass variation of eryngo species worldwide to compare with our findings. However, significant variation has been reported in complexity (total number of shoots per individual), the number of leaves and flowers, and various developmental phase transitions (vegetative to generative and vice versa) of E. maritimum from Southeastern Baltic coast (N56°48′4″, E21°4′4″) (Ievinsh et al., 2020). In a study on the diversity of morphological and biochemical traits of 52 froriepia (Froriepia subpinnata Ledeb. Bail, Apiaceae) accessions from Guilan province (Iran), Jorkesh et al. (2020) reported that leaf and leaflet lengths were the most divers among various parameters, so that the highest variation was observed in leaflet width and leaf number. It has been acknowledged that plant morpho-physiological parameters, chemical constituents, and biological activities are influenced by both environmental and genetic factors (Heywood, 2002). In particular, it was established that photochemical performance of photosynthesis in E. maritimum is extremely sensitive to periods of increased precipitation (Andersone et al. 2011). Also, prolonged periods of precipitation together with low air temperature were devastating for eryngium seeds after ripening, resulting in extremely poor seed germination rate and subsequent seedling growth (Necajeva and Ievinsh 2013). In the present study, the greater biomass accumulation of E. caucasicum from Javaherdeh and Eshkevar sites may be attributed to the higher mean annual rainfall and air temperature compared to the other studied locations (Table 1). In addition, the  Figure 1.
There were significant (positive/and or negative) correlations between some traits as presented with Pearson's correlation general overview and coefficients (Table 3)  and PC7 explained 0.03% and 0.004% of the total variance detected and revealed positive correlations with petiole length and leaf width, respectively (Table 4). In a study on phenotypic di-

| Biochemical and antioxidant evaluations
According to the ANOVA, significant (p < .01) variations were ob-  (Table 2). Moreover, the rest biochemical parameters (rosmarinic acid, total phenolics, and flavonoid contents) showed CVs greater than 50.00%, displaying a high diversity among the studied populations.
Essential oils content varied from 0.011% to 0.30% with an average of 0.28% (Table 2). These values have been in accordance with the previous data reported was as 0.29% in E. amethystinum from Amiata Mount, Italy (Flamini et al., 2008), and 0.06%-0.13% in E. maritimum from Corsica and Sardinia (Darriet et al., 2014). The essential oils yield ranged from 0.0001 to 1.14 g/plant ( Table 2).
Volatiles of the essential oils are largely responsible for the taste of foods. Essential oils of the Eryngium species, even if used in small amounts, its pungent unique odor gives the characteristic flavor to the dishes in which it is incorporated (Flamini et al., 2008). The differences in essential oils content and composition of various Eryngium species and even within the same species may be ascribed to the characteristics of the growth regions (Cardozo et al., 2004;Sefidkon et al., 2004). All these characteristics should be taken into consideration when the plant is used as an aroma source.
Rosmarinic acid content varied from 0.118 to 1.234 μg/g DW with an average of 0.494 μg/g DW (Table 2). In our experiment, a higher content (>0.40 μg/g DW) of this hydroxylated polyphenol was found in most of the individuals and agreed with Kikowska et al. (2022), who reported that rosmarinic acid was the predominant phenolic acid with the highest content in all of the examined tissues of Eryngium spp. in vitro. Also, rosmarinic acid was previously found most notably in other Eryngium species such as E. campestre (Kikowska et al., 2016), E. maritimum (Kikowska et al., 2014), and TA B L E 4 Eigenvectors of principal component axes from PCA for the morphological and biochemical traits and antioxidant activities in the studied eryngo (Eryngium caucasicum Trautv) populations. For the explanation of traits abbreviation, please see Table 2 Trait Component E. planum (Thiem et al., 2013). The range of chicoric acid content was from 0.014 to 0.597 μg/g DW (  (Kremer et al., 2021). The difference might be attributed to ecological situations, edaphic factors, and genetic variations inside the species. HPLC chromatogram of rosmarinic acid and chicoric acid fractions in the leaves of E. caucasicum collected from Javaherdeh is shown in Figure 5.
Total phenolic content varied from 8.85 to 88.15 mg GAE/g extract. Total flavonoid content ranged from 5.41 to 134.40 mg QE/g extract (Table 2). Phenolic acids and flavonoids, precious bioactive secondary metabolites of Eryngium spp., serve a vital role in plants metabolism; their effect, mainly as antioxidants as well as folklore remedies, on human health has been of intense interest to researchers in recent years (Kikowska et al., 2019). Previous studies have revealed the high amounts of total phenolic compounds in different extracts of Eryngium spp., for example, in E. campestre (Guneş et al., 2014;Bouzidi et al., 2017). In addition, the total phenolics and flavonoids content of aqueous, n-hexan, and ethyl acetate extracts in E. caucasicum were reported as 214.18, 29.06, and 140.57 mg GAE/g extract, and 75.36, 97.37, and 31.51 mg/QE g extract, respectively (Nabavi et al., 2012).
In the current study, the antioxidant capacity of the methanol extract of E. caucasicum determined using the free radical DPPH roots parts of E. campestre from Kosovo has previously been evaluated using various testing systems, and findings suggest that, in the DPPH assay, root extract revealed higher radical scavenging activity compared to the extract of the shoot (Nebija et al. 2009).
In a study by Ebrahimzadeh et al. (2009), antioxidant activities of methanol extracts of leaves and inflorescence of E. caucasicum were investigated using six in vitro assay systems, and results showed that leave extract exhibited better Fe 2+ chelating ability than EDTA. Antioxidant molecules and phenolic compounds of plant origin are important to food and pharmaceutical industries due to their unique taste, flavor, and health-promoting properties.
The antioxidant activity of phenolic constituents and flavonoids is basically related to the phenolic rings and hydroxyl groups that enable them to scavenge free radicals, chelate metal ions, and inhibit lipoxygenase activity (Minatel et al., 2017). Here, the antioxidant activity of E. caucasicum might be attributed to their phenolic compounds; rosmarinic acid and chicoric acid were detected in leaf methanol extract ( Figure 5).

F I G U R E 6
Ward cluster analysis of the studied eryngo (Eryngium caucasicum Trautv) populations (Javaherdeh, Kelardasht, Dohezar, Sehezar, Javaherdasht, and Eshkevar represented by black, green, red, blue, aqua, and blueviolet color symbols, respectively) based on the biochemical traits and antioxidant activities by Euclidean distances F I G U R E 7 Two-dimensional scatter plot for PC1 and PC2 based on the biochemical traits and antioxidant activities of the individuals (No.: 1-10) of eryngo (Eryngium caucasicum Trautv) populations (Javaherdeh, Kelardasht, Dohezar, Sehezar, Javaherdasht, and Eshkevar represented by black, green, red, blue, aqua, and blue-violet color symbols, respectively) F I G U R E 8 Biplot for the studied sites of eryngo (Eryngium caucasicum Trautv) populations including Javaherdeh, Kelardasht, Dohezar, Sehezar, Javaherdasht, and Eshkevar based on the morphological and biochemical characters and antioxidant activities activity based on DPPH was positively and significantly (r 0.05 = 0.66) correlated with antioxidant activity results obtained with Fe 2+ chelating assay. The correlation between total phenol content and antioxidant activity has previously been reported by many authors and their results showed a statistically significant correlation between phenol content and antioxidant potential (Johari & Khong 2019;Guo et al., 2021). In addition, the antioxidant activity of essential oils cannot be ignored either (Farias et al., 2020).
The PC1 explained 72.74% of the total variance was represented by essential oil content (9.36), DPPH (0.81), and ferrous ions (Fe 2+ ) chelation (0.57) with significant correlations (Table 4). The PC2 included two traits including essential oil content ( The population analysis of E. caucasicum based on all measured parameters is given in Figure 8. Biplot for the studied areas showed that the studied populations were classified into three groups. The "Dohezar" was placed in the first group and characterized by antioxidant activity with both DPPH and Fe 2+ chelation methods, total flavonoids, and rosmarinic acid content. The second group consisted of three areas from Guilan and Mazandaran provinces and was divided into two subgroups. The first subgroup included "Sehezar," while the second subgroup consisted of "Javaherdeh" and "Eskevar" locations ( Figure 8). Similarly, the HCA showed clustering of studied variables to explore the similarity between observations and/or clusters based on the Euclidean distance coefficient and the average linkage technique (Figure 9). The result can be visualized using heat maps and dendrograms. The characters grouped together to react in a more similar way to the respective populations than those in other clusters. The diversity among populations may be related to natural hybridization/selection phenomenon, sexual reproduction, and human disturbances (Vilà et al., 2000;López-Caamal and Tovar-Sánchez 2014).
Based on overall analyses of all traits, a broad morphological and biochemical diversity was found among the populations, which could be useful in breeding programs and management of genetic resources. Furthermore, the previous studies conducted have shown the existence of genetic diversity and differentiation in Eryngium spp.

F I G U R E 9
Hierarchical cluster analysis (HCA) for the studied populations of eryngo (Eryngium caucasicum Trautv) based on all morphological and biochemical characteristics and antioxidant activities. Red color represents a higher value than the mean for the specific trait in all the studied population, and while blue color shows a lower amount than the mean. For the explanation of traits' abbreviation, please see Table 2 from various countries/regions (Gaudeul et al., 2004;Minasiewicz et al., 2011;Ieviņa et al., 2019).

| CON CLUS IONS
Eryngo (Eryngium caucasicum Trautv) is known for its importance in the field of herbal nutraceuticals. This species is rich in valuable bioactive compounds such as essential oils, phenolic acids, and flavonoids. The plant leaf traits concerning biomass are the most important characteristics to be considered in the agri-food industry. The current study displayed a high diversity among and within populations of E. caucasicum. According to the multivariate analyses of the studied populations, the "Dohezar" was placed in the first group and characterized by antioxidant activity with DPPH and Fe 2+ chelation methods, total flavonoids, and rosmarinic acid contents. The second group consisted of three areas from Guilan and Mazandaran provinces and was divided into two subgroups. The first subgroup included "Sehezar," while the second subgroup consisted of "Javaherdeh" and "Eskevar" locations.
Moreover, Based on the traits related to leaf dimensions and biomass such as leaf length, leaf width, petiole length, the number of leaves per plant, and shoot fresh and dry weights, as well as in terms of biochemical parameters related to antioxidant properties such as essential oils content and yield, total phenolics and flavonoids, rosmarinic acid and chicoric acid contents, and antioxidant activity, eight individuals plant including "Javaherdeh-2", "Javaherdeh-7", "Javaherdeh-10", "Dohezar-4", "Dohezar-5", "Dohezar-6", "Dohezar-9", and "Javaherdasht-4" were superior, therefore, they could be valuable in breeding and field farming programs.

ACK N OWLED G EM ENTS
The authors would like to express their deepest gratitude to Kariman, The Universityof Western Australia, Perth, Australia, for the technical support.

CO N FLI C T O F I NTE R E S T
The authors declare no conflict of interest.